This Program Project Grant brings together seven principal investigators committed to understanding the principles by which RNA molecules fold into their biologically active structures. The proposal comprises four Projects and a Resource Core, all bound together by a central hypothesis: RNA folding can be understood and eventually predicted from a reductionist perspective. RNA looked like a simple messenger in the early days of structural and molecular biology. The last two decades have demonstrated diverse biological, chemical, and physical roles of RNA, and yet our mechanistic understanding remains limited. Deep investigation of the physical behavior of RNA will enable a full description of gene expression and its control. In particular, a predictive and quantitative understanding will catalyz rational RNA engineering at the molecular and systems biology levels and ultimately facilitate rational therapeutic interventions that target or involve RNA. Our component projects build from simple to complex. Project 1 studies the ion atmosphere that surrounds simple helices and uses an integrated experimental-computational feedback loop to develop an understanding of electrostatics, the largest and most general force influencing the behavior and interactions of RNA. Project 2 focuses on helix-junction-helix motifs, the building blocks of RNA tertiary structure. We will develop and apply novel experimental and analytical approaches to fully describe the conformational ensemble for these elements. Project 3 determines the conformational ensembles of RNAs in their unfolded, folded, and intermediate states and defines the energetics of the transitions between these states. We will compare the behaviors of P4-P6 RNA and RNA/protein complexes to the behavior predicted from the properties of their elements, as delineated from Project 2. We thereby directly test the hypothesis that the energetics and behavior of complex RNAs can be understood from the 'sum of their parts', a hypothesis that if true, will lead to a general ability to predict RNA folding and energetics. Projct 4 builds on the reductionist framework developed in the other projects to now address RNA folding kinetics, testing quantitative predictions and developing novel systematic approaches to dissect the influence of co-transcription folding. Together, these studies will uncover basic properties and behaviors that define the physical, chemical, and biological behavior of RNA and catalyze a transformation from a descriptive to quantitative predictive understanding of RNA.